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Assembly and comparative analysis of the complete mitochondrial genome of Echinacanthus longipes (Acanthaceae), endemic to the Sino-Vietnamese karst flora

BMC Genomics volume 26, Article number: 251 (2025) Cite this article

Abstract

Background

Echinacanthus longipes is an endemic species in the Sino-Vietnamese karst flora in the family Acanthaceae. It displays distinctive environmental adaptation characteristics in karst regions. Although it provides an important model for understanding the role of limestone karst in speciation and endemism, the mitochondrial genome (mtDNA) of E. longipes has not been fully characterized.

Results

Here, the mtDNA of E. longipes was successfully assembled as a complex structure in the form of two small circular and three linear molecules with a total length of 810,200 bp. The annotated results revealed 36 protein-coding genes (PCGs), 22 tRNA genes, and three rRNA genes in this mtDNA. Notably, substantial sequence repeats and more tRNAs translocations from the chloroplast to the mtDNA were identified. Among the PCGs of E. longipes, the majority of 401 RNA editing sites were involved in amino acid transitions to hydrophobic sites. The current phylogenetic analysis based on PCGs revealed the evolution of Lamiales and a close relationship between E. longipes and Avicennia marina. However, comparative analyses, including size, structure, GC contents, and genes, reflected the variation in the mitogenomes within Acanthaceae, and the collinearity analysis confirmed the low level of conservation in the genomes of related species in Lamiales. Moreover, the Ka/Ks analysis revealed that negative selection occurred on most PCGs, with the notable exception of ccmB, which underwent positive selection. Interestingly, the ccmB gene had the most protein editing sites.

Conclusions

This study will be invaluable for the mitochondrial study of Acanthaceae. It also provides extensive information for functional genetic and adaptive studies of Echinacanthus in karst regions in the future.

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Introduction

Acanthaceae comprise approximately 191 genera and approximately 4900 species distributed in tropical and subtropical regions [1, 2]. Echinacanthus Nees is a small genus characterized by its axillary or terminal thyrse inflorescence and the anthers with spurred thecae. It consists of four species, E. attenuatus Nees, E. longipes H. S. Lo et D. Fang, E. longzhouensis H. S. Lo, and E. lofouensis (H. Lév.) J. R. I. Wood [3, 4]. Amongst, E. attenuatus is restricted to Bhutan, India, and Nepal in the western Himalayas, and, the other three species are endemic to southern China and northern Vietnam in the Sino-Vietnamese karst flora [5, 6, 7]. Among them, E. longipes is distributed in western Guangxi and southern Yunnan of China, and northern Vietnam, and moreover, it is acaulescent rosette or caulescent herb with purple corollas growing in the damp spaces of limestone hills (Fig. 1). Additionally, it strongly adapts to shallow karst soil with high pH, lower water storage capacity, and high concentrations of magnesium (Mg) and calcium (Ca) [8]. Therefore, E. longipes is a typical limestone species and displays distinctive environmental adaptation characteristics in karst regions. Although it provides an important model for understanding the role of limestone karst in speciation and endemism, the mitochondrial genome of E. longipes remains poorly characterized.

Fig. 1
figure 1

The plant of Echinacanthus longipes and its habitat

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Mitochondria, as cellular organelles within eukaryotic cells, play important roles in energy provision and diverse physiological activities [9, 10]. Using Margulis’s endosymbiosis theory, mitochondria originated from archaea when eukaryotes engulfed bacteria and then evolved into organelles with independent genomes [11, 12]. Unlike nuclear genomes, which are derived from biparental contributions, mitochondrial genomes exhibit an exclusive maternal inheritance pattern [13]. With the development of high-throughput sequencing technologies, the complexity and the variability of plant mitochondrial genomes were revealed [14]. Furthermore, it has been widely utilized for reconstructing phylogenetic relationships and studies of evolutionary biology [15]. Although Acanthaceae is a large family consisting of 4900 species, the complete mtDNA has been sequenced and submitted to National Center for Biotechnology Information (NCBI) for only one species, Avicennia marina (Forssk.) Vierh. (PP908999.1). Thus, the mitochondrial genomes of species within Acanthaceae need to be further studied.

In the present study, the initial complete mtDNA of E. longipes was reported. This is the first comprehensive study of the mtDNA of an Echinacanthus species. The results contribute to the growth of a mitochondrial DNA database specific to the family Acanthaceae, providing crucial genetic data for Echinacanthus species and identifying the genes underlying positive selection in E. longipes.

Results

Characteristics of the mtDNA of E. longipes

Here, the entire mtDNA of E. longipes was obtained. It was assembled as a complex structure in the form of small circular and linear molecules (Fig. 2). Notably, it consists of five contigs named chromosome 1–5 with descending lengths as follows: 592,141 bp, 65,376 bp, 55,262 bp, 53,691 bp and 43,730 bp (Table 1). In total, the length of the mtDNA of E. longipes is 810,200 bp, and the GC content is 42.4%. Sixty-one unique genes are annotated in the mtDNA, including 22 tRNA genes (trnG-UCC, trnS-UGA, trnC-GCA, trnQ-UUG and trnW-CCA with two copies, trnfM-CAU with three copies, trnI-CAU with four copies, and trnP-UGG with five copies), 36 protein-coding genes (nad4, nad9, ccmC, ccmFn, rps12, and rps14 with two copies), and three rRNA genes (Table 2). Annotation of the mtDNA of E. longipes reveals 24 mitochondrial core genes and 12 noncore genes. The core genes include nine NADH dehydrogenase genes (nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7, and nad9), five ATP synthase genes (atp1, atp4, atp6, atp8, and atp9), four cytochrome C biogenesis genes (ccmB, ccmC, ccmFc, and ccmFn), three cytochrome C oxidase genes (cox1, cox2, and cox3), one transport membrane protein-encoding gene (mttB), one maturase gene (matR), and one apocytochrome b gene (cob). The noncore genes include six ribosomal small subunit genes (rps3, rps4, rps10, rps12, rps13, and rps14), four ribosomal large subunit genes (rpl2, rpl5, rpl10, and rpl16), and two succinate dehydrogenase genes (sdh3 and sdh4). Additionally, ten intron-containing genes (nad1, nad2, nad4, nad5, nad7, ccmFc, cox1, cox2, rps3, and rps10) are identified in the mitogenome of E. longipes.

Fig. 2
figure 2

The maps of Echinacanthus longipes mitochondrial genome

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Table 1 The information of E. longipes mitochondrial genome
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Table 2 The mitochondrial genome encoding genes of E. longipes
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Repeat sequence analysis

A total of 160 simple sequence repeat (SSR) loci, including 19 monometric simple sequence repeats (SSRs) (11.88%), 45 dimeric SSRs (28.13%), 23 trimeric SSRs (14.38%), 66 tetrameric SSRs (41.25%), and seven pentameric SSRs (4.38%), were discovered in the mtDNA of E. longipes. However, no hexameric SSRs were found in this study. As a result, the tetrameric and pentameric SSRs contributed the most and the least, respectively. The total SSR loci were spread across the different chromosomes as follows: 121 were distributed on chromosome 1, 18 were distributed on chromosome 2, seven were distributed on chromosome 3, six were distributed on chromosome 4, and eight were distributed on chromosome 5 (Fig. 3). Tetrameric SSRs, which were the most abundant, were distributed on all chromosomes, with 43 on chromosome 1, 11 on chromosome 2, one on chromosome 3, three on chromosome 4, and eight on chromosome 5. The next most abundant type was dimeric SSRs, which were distributed mainly on chromosome 1 (36). Additionally, as shown in Fig. 3 23 trimeric SSRs were identified on chromosome 1 (18), chromosome 2 (four), and chromosome 4 (one). When monometric SSRs were analyzed, almost all were distributed on chromosome 1. Seven pentameric SSRs were split on chromosomes 1 and 2. Moreover, of the detected SSR regions, most of the SSRs (132) were identified in intergenic spaces, whereas 28 SSRs were located in the nad1, nad2, nad4, ccmFC, cob, rps3, rps10, and rrn18 genetic spaces. Interestingly, the majority of the SSRs were especially rich in A or T bases (Supplementary Table S1).

Fig. 3
figure 3

The simple sequence repeats in E. longipes mitochondrial genome

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Mitochondrial genome of E. longipes also contained numerous dispersed repeats, consisting of 102 pairs of forward repeats and 86 pairs of palindromic repeats (Supplementary Table S2). However, neither reverse repeats nor complement repeats were identified. The 40s bp repeats were the most abundant for both types, accounting for more than half of them (119). Moreover, the longest forward repeats and palindromic repeats were all found on chromosome 1, reaching 20,708 and 8,116 bp. The greatest number of dispersed repeats was detected on chromosome 1, with 94 pairs of forward repeats and 84 pairs of palindromic repeats (Fig. 4).

Fig. 4
figure 4

The dispersed repeats in E. longipes mitochondrial genome

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A total of two tandem repeats with matching degrees greater than 95% and lengths spanning 12–16 bp (Table 3) were present in the mtDNA of E. longipes. In addition, they were all detected on chromosome 1.

Table 3 Distribution of the tandem repeats in E. longipes mitochondrial genome
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Protein-coding genes codon usage analysis

The length of the PCGs in E. longipes was 31,969 bp. For the majority of these genes, ATG was the typical start codon, with the exception of GTG for the rpl16 gene and ACG for the nad4L gene. TGA, TAA, and TAG were detected as termination codons. In the complete mtDNA of Echinacanthus, 61 different types of amino acid codons encoding all 20 amino acids were identified. Among all 61 codons, excluding methionine (AUG) and tryptophan (UGG), which exhibited no codon preference (relative synonymous codon usage (RSCU) = 1), 29 codons were used more frequently than expected (RSCU > 1), and 30 codons had RSCU values less than 1 (Supplementary Table S3). The amino acids with the highest frequencies were serine (Ser), arginine (Arg), and leucine (Leu), whereas tryptophan (Trp) and methionine (Met) had the lowest frequencies (Fig. 5).

Fig. 5
figure 5

The relative synonymous codons usage of E. longipes mitochondrial genome. The x axis shows the amino acid, and the bars represent the value of each amino acid codons

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Prediction of RNA editing sites

A total of 401 potential RNA editing sites were identified across the 34 PCGs from E. longipes mtDNA, primarily involving the conversion of C to T (Supplementary Table S4). Notably, ccmB presented the highest frequency of RNA editing sites, totaling 36 edits. Both the mttB and nad4 genes each presented 32 RNA editing sites. In addition, the nad2 gene was associated with 24 RNA editing events. However, atp1, atp8, and atp9 each had only a single RNA editing site (Fig. 6). Furthermore, 68.33% (274) of these RNA editing sites were located at the second site, whereas 28.68% (115) occurred at the first position of the triplet codes. Twelve particular cases were edited at the first and second sites simultaneously, which led to an amino acid change from proline (CCC or CCT) to phenylalanine (TTC or TTT). As a result of all editing, 194 (48.38%) amino acids underwent a transition from the hydrophilic to the hydrophobic state. Meanwhile, 30 (7.48%) amino acids were anticipated to shift from the hydrophobic to the hydrophilic state. It was also found that two hydrophilic amino acids, arginine and glutamine, were converted to stop codons (Table 4).

Fig. 6
figure 6

The distribution of RNA editing sites of the protein-coding genes in E. longipes mitochondrial genome

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Table 4 Prediction of RNA editing sites in E. longipes
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Homology analysis of genome sequences

The complete chloroplast genome (cpDNA) of E. longipes was 152,644 bp in length, encompassing 113 unique genes [7]. The migration of sequences between the chloroplast and the mitochondria in E. longipes were analyzed. Cumulatively, 72 sequences, varying in length from 71 to 10,303 bp, were identified between the mtDNA and cpDNA (Supplementary Table S5). The total length of these migrant sequences was 118,235 bp, constituting 14.59% of the overall mtDNA. Furthermore, the most frequent migration occurred between chromosome 1 and cpDNA. However, only four sequences migrated between chromosome 2 and cpDNA (Fig. 7). Additionally, annotation of these sequences in mtDNA resulted in the identification of 13 tRNAs, namely, trnD-GUC, trnH-GUG, trnI-GAU, trnL-UAG, trnM-CAU, trnfM-CAU, trnN-GUU, trnP-UGG, trnQ-UUG, trnW-CCA, trnS-GGA, trnS-UGA and trnV-GAC, two PCGs including rps14 and rps12, and one rRNA rrn18 (Supplementary Table S5).

Fig. 7
figure 7

Homology analysis between mitochondrial genome and chloroplast genome of E. longipes. The orange and the green arcs denote the mtDNA and cpDNA, respectively. The blue lines signify the homologous sequences

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Phylogenetic and Ka/Ks analysis

Thirty-one conserved PCGs, including atp1, atp4, atp6, atp8, atp9, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7, nad9, cox1, cox2, cox3, ccmC, ccmB, ccmFC, ccmFN, matR, mttB, rps3, rps4, rps12, rps13, rps14, rpl5, rpl10, and sdh4, which were retrieved from 25 Lamiales species and two outgroup species, were used to construct a phylogenetic tree. As shown in Fig. 8A, all the sampled species from the eight families in the order Lamiales were clustered into one clade with 1.00 posterior probability (PP). The results of this study show that, within Lamiales, Lamiaceae and Orobanchaceae are formed a clade, which was identified as a sister to Lentibulariaceae with a high support value (PP = 1.00). Two species of Acanthaceae, E. longipes and A. marina, are clustered as a group with 1.00 posterior probability value. Therefore, E. longipes was determined to be closely related to A. marina in the present study. Acanthaceae are formed a sister group to the branch containing Lamiaceae, Orobanchaceae, Lentibulariaceae and Bignoniaceae, and the analyses obtained high support for the node (PP = 1.00). At the same time, the present phylogenetic tree is favored Plantaginaceae and the branch of the above families including Lamiaceae, Orobanchaceae, Lentibulariaceae, Bignoniaceae and Acanthaceaeas as a sister group. Additionally, Oleaceae is the earliest diverging lineage in Lamiales in this study.

Fig. 8
figure 8

Phylogeny and Ka/Ks analysis of eight families in Lamiales. (A) Phylogenic tree of eight families in Lamiales based on 31 PCGs. The numbers of the branches represent posterior probability (PP). (B) The Ka/Ks values of 16 protein-coding genes of E. longipes versus 24 species of Lamiales

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The rates of nonsynonymous substitutions (Ka) and synonymous substitutions (Ks) were calculated to evaluate whether selective pressure existed on PCGs during evolution within Lamiales (Supplementary Table S6). When the Ka/Ks ratio is > 1, genes are undergoing positive selection, conversely, Ka/Ks < 1 denotes negative selection, and Ka/Ks = 1 represents neutral selection. Thus, 16 PCGs from E. longipes mtDNA were compared with those from the selected species in Lamiales. The Ka/Ks values of most PCGs in E. longipes were determined to be less than 1. The genes with Ka/Ks values higher than 1 included ccmB, ccmC, cox3, matR and so on. However, ccmB was the only gene with Ka/Ks > 1 compared with all the other species (Fig. 8B).

Comparison of the mtDNA of E. longipes and other species

Firstly, the comparative mtDNA analysis will be performed among species within Acanthaceae. To date, the available mitochondrial information in Acanthaceae is limited to A. marina. So, the comparisons will be focus on E. longipes and A. marina. As shown in Table 5, the differences between A. marina and E. longipes are reflected mainly in the size, structures, GC contents and genes. Combination the data of E. longipes in this study and the data of A. marina in NCBI (https://www.ncbi.nlm.nih.gov/nuccore/PP908999.1?report=genbank), a deeply comparison of these mitochondrial genomes was conducted. The mtDNA of A. marina is typically single circular, in contrast, E. longipes possesses complex structures with three linear and two circular chromosomes. The gene annotations of these two genomes were compared with each other. Notably, the mtDNA of E. longipes lost rps7, but it had more tRNA genetic duplications which may lead to their differences in length.

Table 5 General features of the two mitochondrial genomes in Acanthaceae
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In order to illustrate the homologous of mitochondrial genomes between E. longipes and the closely related species in Lamiales, E. longipes was further compared with A. marina (Acanthaceae), Plantoga ovata (Plantaginaceae), Markhamia cauda-felina (Bignoniaceae) and Utricularia reniformis (Lentibulariaceae) using the Blastn program to analyze the homologous collinear blocks, excluding blocks less than 0.5 kb in length. As a result, some fine homologous regions and numerous inversion regions were identified between E. longipes and the related species. However, the arrangement order of collinear blocks was inconsistent across these species. Additionally, compared with other species, unique regions with substantial genomic rearrangements were identified in the mtDNA of E. longipes (Fig. 9).

Fig. 9
figure 9

Collinear analysis across the mitochondrial genomes of E. longipes, A. marina and three other closed species in Lamiales. The gray ribbons indicate fine homologous regions and the pink ribbons indicate the inversion regions

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Discussion

Mitochondria are the “energy factories” within eukaryotic cells. In addition, they also play critical roles in physiological activities such as information transmission, division, cell differentiation, and apoptosis [9, 10]. In 1992, the first mitochondrial genome from terrestrial plants was reported [16]. An increasing number of plants mitochondrial genome have since been sequenced and reported. Acanthaceae are a large family of angiosperms comprising approximately 191 genera and 4900 species [2]. However, until now, only one mtDNA within Acanthaceae has been reported (submitted to NCBI). In the present study, the complete mtDNA of E. longipes, an endemic species in the Sino-Vietnamese karst region, was sequenced and assembled. It was the first mtDNA sequenced from the genus Echinacanthus. According to previous studies, the length of angiosperm mitochondria genomes usually ranges from 221 kb to 11.3 mb [10]. The mtDNA of E. longipes spans 810,200 bp, with 61 genes encompassing 36 PCGs, 22 tRNA genes and three rRNA genes. In addition to their typical circular shape, the mitochondrial genomes of terrestrial plants present complex structures, featuring linear and branched molecules. In contrast to previous studies, in which the predominant structures of plant mitochondrial genomes including the single circular (e.g., Suaeda glauca and Rotheca serrata), multiple circulars (e.g., Angelica biserrate and Ajuga ciliata), linear (e.g., Primulina hunanensis) and branched (e.g., Ventilago leiocarpa and Quercus acutissima) configurations [17-22], the E. longipes mtDNA exhibits a complex structure consisting of three linear and two circular chromosomes.

Repeat sequences are considered to play a vital role in shaping the mitochondrial genomes because of intermolecular recombination [17]. They are widely dispersed in the mitochondrial genome and have been extensively used in biological evolution and population genetics studies [23]. In the present study, three types of repeat sequences, SSRs, dispersed repeats and tandem repeats, were identified across the mtDNA of E. longipes. The majority of SSRs were tetrameric repeats. These tetrameric repeats could serve as candidate molecular markers in Echinacanthus for future research on species identification, speciation mechanisms, and interspecific polymorphisms. Moreover, the SSRs in E. longipes were mainly located in intergenic spacer regions and contributed to A/T richness. Consistent with observations in other angiosperms such as Primulina hunanensis, Gleditsia sinensis and Acer truncatum, the elevated AT-content in SSR regions further supports the genome-wide high AT composition observed in plant mitochondrial genomes [18, 24, 25]. Notably, compared with two tandem repeats, dispersed repeats including forward and palindromic repeats, accounted for 188 in the E. longipes mtDNA. These highly repetitive sequences may not only increase the frequency of intermolecular recombination but also change the conformations and sizes of plant mitochondrial genomes [17, 18, 26-29].

Thirty-six PCGs with a total length of 31,969 bp were aligned in the E. longipes mtDNA. The analysis of codon usage in E. longipes presented herein indicates that mtDNA predominantly utilizes ATG as the initiation codon. Additionally, GTG serves as translation initiation codon for the rpl16 gene in plant mtDNA [30]. Furthermore, RNA editing, an RNA nucleotide posttranscriptional modification process, widely occurs in the PCGS of mitochondrial genomes and significantly affects gene expression [17, 31]. Previous reports have shown that the number of editing sites and the genes with the maximum number of editing sites vary across species [32]. Here, a total of 401 RNA editing sites within 34 PCGs were identified, and ccmB presented the greatest number of RNA editing sites. Most RNA editing events involved the conversion to hydrophobic amino acids in the mtDNA of E. longipes, which is helpful for altering physicochemical properties and increasing protein folding [31, 33, 34]. Another remarkable variation among these RNA editing sites featured hydrophilic amino acids changing to stop codons in the rps10 and atp6 genes, which may provide essential clues for nonfunctional editing and the consideration of pseudogenes in E. longipes [22, 35].

The phylogenetic relationships of E. longipes with other related species in Lamiales based on PCGs of the mitochondrial genomes were analyzed. Phylogenetic analyses of eight families confirmed the monophyly of Lamiales. Notably, the current phylogenetic relationships within Lamiales based on PCGs of the mitochondrial genomes were the same as those reported by APGIV [36]. Furthermore, E. longipes was determined to be closely related to A. marina in Acanthaceae. To further explore the evolutionary characteristics of the mitochondrial genomes within Acanthaceae, we exhaustively compared E. longipes with A. marina. Notably, the present study revealed relatively conserved GC contents but considerable structural variation in their mitochondrial genomes. And the different genome components exhibited different lengths ranging from 574,037 bp to 810,200 bp. Moreover, the core gene contents in the mitochondrial genomes of these two Acanthaceae species remained relatively consistent, but the numbers of noncore genes and tRNA genes copies varied significantly. Colinear analysis plays an important role in revealing species evolution and diversity [18, 37]. Therefore, further investigations of collinearity among the mitochondrial genomes of E. longipes, A. marina and three related species in Lamiales were conducted to determine their organization. The results revealed that these mitochondrial genomes exhibited substantial rearrangement and limited collinearity, which was considered a driving force for the mtDNA evolution of E. longipes in Lamiales. Thus, the results of the current phylogenetic and comparative analyses suggest that mitochondrial genomes are useful for studying the phylogeny, evolution and speciation.

Acanthaceae are a large family of angiosperms with high species, geographic, and ecological diversity [2, 38]. Echinacanthus longipes is a species endemic to karst regions. Therefore, it is necessary to analyze the adaptive evolution of genes in E. longipes. The nonsynonymous substitution and synonymous substitution ratio (Ka/Ks) are very useful for measuring selective pressure at the protein level in mtDNA [39]. Genes with positive selection play key roles in adaptation to diverse environments [7, 40]. Ka/Ks analysis of PCGs between E. longipes and other Lamiales species was carried out. As a result, most of the PCG genes with Ka/Ks values < 1 underwent negative selection. However, only one gene, ccmB, with Ka/Ks > 1 had undergone positive selection compared with all the other selected species. Therefore, the ccmB gene, which plays an important role in cytochrome c biogenesis, was determined to be under positive pressure in E. longipes. Interestingly, this gene is also subjected to positive selection in Suaeda glauca, which is a prominent salt-tolerant species [17]. Notably, the ccmB gene had the most protein editing sites in mtDNA of E. longipes. A further study of the gene ccmB was necessary in the future.

Mitochondrial plastid DNAs (MTPTs), which are sequences transferred from plastid (e.g., chloroplast) to mtDNA, are common in angiosperms and originated at least 300 million years ago (Mya) [41, 42]. It was originally hypothesized that majority of the MTPTs usually became nonfunctional genes [43]. The lengths of MTPTs vary significantly across different species, ranging from less than 1 kb to more than 130 kb, and constitute 1–12% of mtDNA [18, 22, 44]. In the present analysis, E. longipes presented 118,235 bp MTPTs, accounting for 14.59% of the mtDNA. These results indicate that there were more transfer events in E. longipes than that in most reported plants. Moreover, the cpDNA of E. longipes contributed numerous sequences to its mtDNA, which may explain the complicated structure and diversity of the mtDNA of E. longipes. These MTPTs included tRNA genes, protein-coding genes, intergenic regions, and even part of the rRNA gene. Notably, tRNA genes exhibited the highest frequency of MTPTs in E. longipes, a pattern consistent with prior observations in other species (e.g., Capsicum pubescens and Astragalus membranaceus) [45, 46]. In addition to the common tRNA genes in angiosperms, namely, trnH-GUG, trnM-CAU, trnN-GUU, trnW-CCA, trnP-UGG, trnS-GGA, and trnD-GUC (in dicots), five genes, trnI-GAU, trnL-UAG, trnfM-CAU, trnS-UGA, and trnV-GAC, were identified in the MTPTs of E. longipes. Previous studies demonstrate that the transfer of a greater number of tRNA genes from chloroplasts to mitochondria is more conducive to meeting the demands of protein synthesis, thereby facilitating plant evolution and adaptation [34, 47]. Thus, in E. longipes, the increased number of tRNA genes associated with MTPTs may increase adaptation to karst environments. However, the underlying mechanism and role of MTPTs deserve in-depth study in the future.

Conclusions

This study successfully assembled and annotated the complete mtDNA of E. longipes, an endemic species in the Sino-Vietnamese karst region, for the first time. It spans 810,200 bp, with 61 unique genes encompassing 36 PCGs, 22 tRNA genes and three rRNA genes, and displays a complex structure in the form of two small circular chromosomes and three linear chromosomes. Two tandem repeats, 188 dispersed repeats and 160 simple sequence repeats were identified in the E. longipes mtDNA. Moreover, 401 RNA editing sites indicated that ccmB had the most protein editing sites and the most amino acids were converted to hydrophobic residues. Furthermore, this research confirmed that more MTPTs occurred in the tRNA genes of E. longipes. The results of the current phylogenetic and comparative analyses raise the possibility that mitochondrial genomes are useful for studying the phylogeny and speciation of Lamiales. Ka/Ks analysis revealed that most of the genes underwent negative selection. However, ccmB is the only gene that has undergone positive selection when compared with all the other selected species. This study will be invaluable for the mitochondrial study of Acanthaceae. It also provides extensive information for functional genetic and adaptive studies of Echinacanthus in karst regions in the future.

Materials and methods

Plant materials, DNA extraction and sequencing

Plant sample was investigated and collected by Yusong Huang, Yunfei Deng, Yi Tong et al. in its native habitats from Jingxi County, Guangxi, China. This area is a public land and permitted to collect E. longipes. The sample was identified by Yunfei Deng and the voucher specimen (No.16101704) was deposited in Herbarium of South China Botanical Garden, Chinese Academy of Sciences (IBSC). Total genomic DNA was extracted from 100 mg silica gel-dried leaves following the method of CTAB [48]. After removing RNA contaminants and checking the quality of the DNA, an excellent integrity of DNA molecules was observed. The library construction and sequencing procedures were performed at Biomarker Technologies CO., LTD (Beijing, China). For PacBio HiFi sequencing, genomic DNA was fragmented to 15 kb to construct a long-read library (SMRT bell library) according to the manufacturer’s instructions (Pacific Biosciences, CA, USA), and then the library was sequenced on a PacBio Sequel II platform. After filtering out the low-quality reads and sequence adapters, 23.69Gb of clean reads with N50 = 13.51Kb were obtained.

Assembly and annotation of mtDNA

The PacBio HiFi sequencing data was used to assemble the mtDNA. Mitochondrial genomes of three species, including Liriodendron tulipifera L. (NC_021152.1), Ajuga reptans L. (NC_023103.1), and Rotheca serrata (L.) Steane & Mabb. (NC_049064.1) were downloaded from GenBank database as reference sequences. The Seqkit software was used to select 10X contigs as mitochondrial sequences [49]. Then, the assembly was performed using Flye software with the default parameters [50]. Bandage software was used to check the result of the assembly and screen the mitochondrial sequences again [51]. Finally, the complete mtDNA was obtained by the comparison results of blast. The mitogenomes were annotated by the online tool IPMGA (http://www.1kmpg.cn/ipmga/) using L. tulipifera, A. reptans, and R. serrata as reference genomes and visualized in the online tool OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) [52, 53].

Repeat sequence analysis

The simple sequence repeats of E. longipes were identified with MISA by setting the minimum number of repeats to 10, 5, 4, 3, 3 and 3 for mono-, di-, tri-, tetra-, penta- and hexanucleotides, respectively [54]. The online tool REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer) was used to analyze dispersed repeats with settings as follows: 3 for hamming distance, 40 for minimal repeat size, and 5000 for maximum computed repeats. Tandem repeats were obtained by TRF network server (https://tandem.bu.edu/trf/trf.html).

Codon usage analysis

The PCGs were extracted using Phylosuite software with default settings [55]. MEGA v7.0 software was employed to analyze codon usage bias through calculation of RSCU values based on the PCGs of the mtDNA [56].

Prediction of RNA editing sites

The RNA editing sites from all PCGs encoded within the mtDNA of E. longipes were predicted on the Plant Predictive RNA Editor (PREP) suite (http://prep.unl.edu/) with a cut off value of 0.2.

Homology analysis

The cpDNA of E. longipes (NC_039761) was downloaded from NCBI Organelle Genome Resources Database. Identification of genes transferred from chloroplasts to mitochondria were detected using BLASTN software on NCBI with parameters “e-value ≤ 1e− 10, length ≥ 50, matching rate ≥ 90%”, and the outcomes were visually represented utilizing the advanced circos package in TBtools [57].

Phylogenomic and Ka/Ks analysis

Twenty-five mitochondria genomes of eight families in Lamiales and two outgroups were downloaded from GenBank. Thirty-one conserved protein-coding genes of all mitochondria genomes were extracted in Phylosuite software and aligned with MAFFT v.7 (auto-strategy) [58]. TVM + I + G model was selected as the best model using program Modeltest 3.7 [59]. Then, the concatenated data matrix of the PCGs was subjected to Bayesian analysis using MrBayes v.3.2 [60]. All parameters were set according to the TVM + I + G model as follows: statefreqpr = fixed (0.2604, 0.2130, 0.2118, 0.3149) revmat = fixed (1.4764, 1.5241, 0.5723, 0.9977, 1.5241, 1.000), shapepr = fixed (0.7854), pinvar = fixed (0.4345). The analysis implemented Markov chain Monte Carlo (MCMC) algorithm and ran for 1,000,000 generations. The first 1,000 trees were considered as the “burn-in” period and discarded. The remaining trees were used to construct the majority-rule consensus tree. Posterior probabilities > 0.95 were considered significant support for a clade. Meanwhile, the Ka and Ks rates of the 16 PCGs in E. longipes with the 24 species of Lamiales were analyzed in DnaSP v.6 [61]. And then ratios of Ka/Ks were calculated.

Colinear analysis

The comparisons between the mtDNA of E. longipes and four closely related species were conducted in Blastn 2.14.0 + software with setting as “-evalue 1e-5 -outfmt 6”. Then the MCScanX software and Mauve 2.4.0 were used to generate the multiple synteny plot and analysis of mitogenome collinearity [62, 63].

Data availability

The accession number of Echinacanthus longipes mitogenome in Gene Bank (NCBI) is PQ164709- PQ164713. All data generated or analyzed during this study were provided as supplementary information files.

Abbreviations

NCBI:

National Center for Biotechnology Information.

PCGs:

Protein-coding genes.

RSCU:

Relative synonymous codon usage.

SSRs:

Simple sequence repeats.

cpDNA:

Chloroplast genome.

mtDNA:

Mitochondrial genome.

MTPTs:

Mitochondrial plastid DNAs.

Ka:

Nonsynonymous substitutions.

Ks:

Synonymous substitutions.

PP:

Posterior probability.

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Acknowledgements

We are grateful to Mr. Yi Tong and Ms. Chunyu Zou for their help in taking photos of Echinacanthus longipes.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos. 32070363, 31400184, 32470219), and Southeast Asia Biodiversity Research Institute, the Chinese Academy of Sciences (Y4ZK111B01).

Author information

Authors and Affiliations

  1. College of Biological and Environmental Engineering, Shandong University of Aeronautics, Binzhou, 256600, China

    Chunming Gao

  2. State Key Laboratory of Plant Diversity and Specialty Crops, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong, 510650, China

    Shu Wang, Yusong Huang & Yunfei Deng

  3. Key Laboratory of National Forestry and Grassland Administration on Plant Conservation and Utilization in Southern China, Guangzhou, 510650, China

    Shu Wang, Yusong Huang & Yunfei Deng

  4. University of the Chinese Academy of Sciences, Beijing, 100049, China

    Shu Wang & Yusong Huang

  5. Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and the Chinese Academy of Sciences, Guilin, Guangxi, 541006, China

    Yusong Huang

  6. Southeast Asia Bioaffiliationersity Research Institute, Chinese Academy of Sciences, Yezin, Nay Pyi Taw, 05282, Myanmar

    Yunfei Deng

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  1. Chunming Gao
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Contributions

CMG designed the project, analyzed the data and draft this paper. SW assembled and annotated the mitogenome. YSH investigated and collected the samples. YFD conceived and revised this paper. All authors read the manuscript.

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Correspondence to Yunfei Deng.

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Gao, C., Wang, S., Huang, Y. et al. Assembly and comparative analysis of the complete mitochondrial genome of Echinacanthus longipes (Acanthaceae), endemic to the Sino-Vietnamese karst flora. BMC Genomics 26, 251 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11448-6

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BMC Genomics

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